Discussion

The buffering capacity of NSCs in response to the late frost
The seasonal dynamics of NSCs play a crucial role in regulating C source-sink balance through buffering the difference between C supply and demand (Scartazza et al. 2013; Fatichi et al. 2014; Collalti et al. 2018, 2020). The complete destruction of photosynthetic apparatus and the consequent strong reduction of stem radial growth during springtime 2016 (May-June), which in diffuse-porous tree species is mainly supplied by the new assimilates (Barbaroux & Bréda 2002; Čufar, Prislan, De Luis & Gričar 2008; Zein et al.2011; Michelot, Simard, Rathgeber, Dufrêne & Damesin 2012), were associated with an increase of stemwood NSCs due to starch accumulation. An increase in total stemwood NSCs has been previously observed from November to March in other temperate forests and it was attributed to remobilization of sugars from storage compartments in coarse roots in advance of the C demands associated with springtime growth (Hoch, Richter & Körner 2003; Hartmann & Trumbore 2016). The NSCs seasonal dynamic shows that starch accumulation in beech occurs during the formation of the new crown, in the presence of the potentially dominating sink represented by new growing leaves and shoots, while soluble sugars are decreasing. Our results confirm that the accumulation of starch in stemwood of beech trees during springtime is not necessarily supported by freshly produced photosynthates. In 2016, it occurs, uniquely, as the result of the remobilization of already existing soluble NSCs, including those remobilised from below-ground organs. The normal starch rise in spring could be favoured by the destruction of the developing canopy leaves. This condition leads to a high concentration of soluble sugars within the stemwood that, concurrently to the springtime increased air temperatures, favour synthesis of starch over its degradation (Witt & Sauter 1994). Indeed, it was recently demonstrated in one-year old shoots of Juglans regia L. that wood accumulation of starch, accompanied by an increase of the total activity of ADPglucose pyrophosphorylase, can be increased when photosynthate export from the shoot is blocked by girdling (Moscatello et al. 2017). Thus, the spring programmed activation of starch synthesis in wood can occur even when C resources are very limited by the absence of a photosynthesizing crown. This strongly supports the, much debated, hypothesis of an active control of the accumulation and buffering role of NSCs in wood (Sala et al.2012; Collalti et al. 2020).
The buffering key role of NSCs to compensate the difference between C sink and C supply was also particularly evident during the late spring and early summer 2016, when stemwood starch reserves were partially hydrolysed, and soluble non-structural carbon became increasingly older (D’Andreaet al. 2019), for sustaining the second leaf re-sprouting and other plants needs, as cambium activity (D’Andrea et al. 2020b). Usually, during the second part of the season (August-September), the new assimilates from the canopy are mainly used to sustain C sink activities related to wall thickening and lignification phase (Prislan, Čufar, De Luis & Gričar 2018) and to refill the starch reserves within the stemwood. However, after the “second” green up in July 2016, C allocation to cell wall thickening, was extremely limited due to strong reduction of xylem cells production (D’Andrea et al. 2020b) leading to the increase of both starch and soluble sugars in stemwood of beech plants at the end of August. The reduced sink activity (related to radial growth, wall thickening and lignification) during extreme weather events could be functional to prevent NSCs depletion (Anderegg 2012; Dietrich, Delzon, Hoch & Kahmen 2018). It should be noted that NSCs, including starch, can be rapidly transformed, ensuring a rapid hexose supply to the hexose phosphate pool. The hexose phosphate pool then supports both metabolic and structural cell requirements for reduced carbon, ranging from glycolysis and respiratory metabolism to cell wall polymer synthesis. On the contrary, assimilates ending up in cell wall components cannot be used for metabolism, because compartmentalized and sequestered away from sites of phloem loading, potential dead alley for their usage. Hence, under photosynthate famine and starving conditions, prioritization of photosynthates allocation to NSCs over any other form of growth, might ensure the maintenance of a sufficient amount of metabolically available level of carbon reserves. This allocation choice reflects a more conservative, and less costly, strategy than supporting end point-like allocation of photosynthates to cell wall components and ensures a much higher plasticity to sustain plant response to environmental constraints (Rodríguez-Calcerrada et al. 2019; Collalti et al . 2020).
In 2016 year, the strong reduction of sink activity, concomitantly with the seasonal decrease of air temperatures, could contribute to the slightly anticipated closure of the season. After leaf shedding, starch was partly hydrolysed and converted to soluble sugars to reduce cell osmotic potential and induce cold tolerance (Bonhomme, Rageau, Lacointe & Gendraud 2005; Tixier & Sperling 2015).
The summer drought effects on growth and NSCs
In 2017, at the beginning of vegetative season, the new assimilates produced by the canopy photosynthesis were mainly used for sustaining the stem radial growth, which, differently from 2016, reached similar values of BAI to those observed for the reference period (1989-2015). In 2017, the summer drought affected NSCs dynamic while had only very limited effects on annual stem radial growth. Such a behaviour has been already observed for other tree species growing in the Mediterranean area which adopt a stress avoidance strategy, adjusting the end of xylem growth before potential stressful conditions may occur (e.g. Lempereuret al. 2015; Forner, Valladares, Bonal, Granier & Grossiord 2018).
The slight reduction of C reserves at the end of the 2016 growing season impacted the dynamic of the following year. Notwithstanding that the content of starch showed the typical seasonal trend of the site, the starch and total sugars content in woody tissue from bud break till the end of June 2017 was clearly lower than the modelled reference NSCs dynamic of the site. In summer 2017, the warm drought event had a strong effect on NSCs dynamic, leading to starch hydrolysis and accumulation of soluble sugars in woody tissue. As drought induces a partial stomatal closure that reduces C uptake, trees depend more on NSCs storage to sustain metabolic activities, defence mechanisms against pathogens and osmoregulation processes (McDowell 2011; Hartmann & Trumbore 2016; Collalti et al . 2020). The observed increase of wood soluble sugars concentration during July-August 2017 is in agreement with the key role of these non-structural compounds as solutes for osmoregulation (Chaves, Maroco & Pereira 2003). Plants under drought conditions can actively control the osmotic cell pressure to avoid tissue dehydration and to maintain the physiological functions by increasing the concentration of different kinds of compatible solutes such as betaines, amino acids and sugars (Morgan 1984). In our study, the increased concentration of stemwood soluble sugars during drought was due to both hexoses (glucose and fructose) and sucrose (data not shown), according to previous findings (Fu & Fry 2010; Yang 2013). In addition, NSCs have also a relevant role to maintain xylem transport and embolism repair under drought conditions (Scartazza, Moscatello, Matteucci, Battistelli & Brugnoli 2015; Hartmann & Trumbore 2016). The so called ‘C starvation hypothesis ’ (Mcdowell et al. 2008) speculates that the drought-induced stomatal closure minimizes hydraulic failure but, at the same time, causes a decline of photosynthetic uptake, possibly leading to C starvation as carbohydrates demand continues for the maintenance of metabolism and defence. In addition, the concomitance of elevated temperatures could accelerate metabolism and, thus, the starch depletion potentially leading to tree mortality (Adams et al.2009), suggesting that trees, to avoid this risk, should be able to maintain a minimum (safety) level of reserve under drought and warm conditions (McDowell & Sevanto 2010). Our results support this hypothesis, showing that, notwithstanding the partial starch hydrolysis, the total NSCs contents were only slightly affected, indicating that at least beech trees are able to counteract a relatively brief and intense hot drought event by the interconversion between starch and soluble sugars without drastically affecting the total C storage reserves in woody tissue. However, at the end of the 2017 vegetative season, trees were able to store similar amounts of starch and total NSCs compared to the modelled reference value of the site, confirming that the studied forest showed an efficient internal regulation mechanism able to respond resiliently to environmental factors with short- to medium-term homeostatic equilibrium (Scartazza et al. 2013; Dietrich et al. 2018). The absence of a strong depletion of NSCs at the end of two sequential years characterised by extreme weather events that strongly reduced C supply and, at the same time, increased C demand for sustaining stress-recovery (frost) and stress-tolerance (drought) processes, further support the hypothesis that C reserves in plants can be tightly actively managed. In this view, wood NCS synthesis, cleavage, interconversion, mobilisation and allocation need to be actively controlled at the physiological biochemical and molecular level, to optimize growth and survival in the long-term (Sala et al. 2012; Collalti et al . 2018; Merganikova et al . 2019; Collaltiet al. 2020).
Summarizing, our study gives some new insights while elucidating the mechanisms connected to the impact of late frost and summer drought on sink processes (stem and foliage growth, allocation to reserve pool) in a Mediterranean beech forest. Synthesis, cleavage, interconversion, mobilisation and allocation of wood NSCs are all finely regulated processes and play a key role in counteracting the negative effects of both late frost and summer drought, ensuring plant survival and buffering the difference between C supply and demand under extreme weather event conditions. This information suggests that C reserves could be crucial for resilience of beech, but not only, because of the expected increasing frequency of extreme weather events under the future global changes and may be useful for adaptive future management strategies of forests in the Mediterranean area and Europe.
Acknowledgements
Collelongo-Selva Piana is one of the sites of the Italian Long-Term Ecological Research network (LTER-Italy), part of the International LTER network (ILTER). Research at the site in the years of this study was funded by the eLTER H2020 project (grant agreement no. 654359). Activity and data analysis at the site are currently funded by resources available from the Italian Ministry of University and Research (FOE-2019), under projects Climate Changes (CNR DTA. AD003.474) and Green & Circular Economy—GECE (CNR DBA. AD003.139). The